The present invention relates to novel benzamides which are inhibitors of enzymes, especially cysteine proteases such as calpain (=calcium dependant cysteine proteases) and its isoenzymes and cathepsins, for example B and L.
Calpains are intracellular proteolytic enzymes from the group of cysteine proteases and are found in many cells. Calpains are activated by an increase in the calcium concentration, a distinction being made between calpain I or xcexc-calpain, which is activated by xcexc-molar concentrations of calcium ions, and calpain II or m-calpain, which is activated by m-molar concentrations of calcium ions (P. Johnson, Int. J. Biochem. 1990, 22(8), 811-22). Further calpain isoenzymes have now been postulated too (K. Suzuki et al., Biol. Chem. Hoppe-Seyler, 1995, 367(9), 523-9).
It is suspected that calpains play an important part in various physiological processes. These include cleavages of regulatory proteins such as protein kinase C, cytoskeletal proteins such as MAP 2 and spectrin, muscle proteins, protein degradation in rheumatoid arthritis, proteins in the activation of platelets, neuropeptide metabolism, proteins in mitosis and others which are listed in M. J. Barrett et al., Life Sci. 1991, 48, 1659-69 and K. K. Wang et al., Trends in Pharmacol. Sci., 1994, 15, 412-9.
Elevated calpain levels have been measured in various pathophysiological processes, for example: ischemia of the heart (e.g. myocardial infarct), of the kidney or of the central nervous system (e.g. stroke), inflammations, muscular dystrophies, cataracts of the eyes, injuries to the central nervous system (e.g. trauma), Alzheimer""s disease etc. (see K. K. Wang, above). It is suspected that there is a connection between these disorders and elevated and persistent intracellular calcium levels. This results in overactivation of calcium-dependent processes, which are then no longer subject to physiological control. Accordingly, overactivation of calpains may also induce pathophysiological processes.
It has therefore been postulated that inhibitors of calpain enzymes may be useful for treating these disorders. Various investigations have confirmed this. Thus, Seung-Chyul Hong et al., Stroke 1994, 25(3), 663-9 and R. T. Bartus et al., Neurological Res. 1995, 17, 249-58 have shown a neuroprotective effect of calpain inhibitors in acute neurodegenerative disorders or ischemias like those occurring after stroke. Likewise, calpain inhibitors improved the recovery of the memory deficits and neuromotor disturbances occurring after experimental brain trauma (K. E. Saatman et al. Proc. Natl. Acad. Sci. USA, 1996, 93, 3428-3433). C. L. Edelstein et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 7662-6, found a protective effect of calpain inhibitors on kidneys damaged by hypoxia. Yoshida, Ken Ischi et al., Jap. Circ. J. 1995, 59(1), 40-8, were able to show beneficial effects of calpain inhibitors after cardiac damage produced by ischemia or reperfusion. Since the release of the xcex2-AP4 protein is inhibited by calpain inhibitors, a potential therapeutic use for Alzheimer""s disease has been proposed (J. Higaki et al., Neuron, 1995, 14, 651-59). The release of interleukin-1xcex1 is likewise inhibited by calpain inhibitors (N. Watanabe et al., Cytokine 1994, 6(6), 597-601). It has further been found that calpain inhibitors have cytotoxic effects on tumor cells (E. Shiba et al. 20th Meeting Int. Ass. Breast Cancer Res., Sendai Jp, Sep. 25 to 28, 1994 Intl. J. Oncol. 5 (Suppl.), 1994, 381). Further possible uses of calpain inhibitors are detailed in K. K. Wang, Trends in Pharmacol. Sci., 1994, 15, 412-8.
Calpain inhibitors have already been described in the literature. However, these are mainly peptide inhibitors. Many known reversible inhibitors of cysteine proteases such as calpain are, however, peptide aldehydes, in particular dipeptide and tripepide [sic] aldehydes such as, for example, Z-Val-Phe-H (MDL 28170) (S. Mehdi, Trends in Biol. Sci. 1991, 16, 150-3). Under physiological conditions, peptide aldehydes have the disadvantage, owing to their great reactivity, that they are often unstable, may be rapidly metabolized and are prone to nonspecific reactions which may cause toxic effects (J. A. Fehrentz and B. Castro, Synthesis 1983, 676-78).
Peptide ketone derivatives are likewise inhibitors of cysteine proteases, in particular calpains. Thus, for example, ketone derivatives where the keto group is activated by an electron-attracting group such as CF3 are known to be inhibitors of serine proteases. In the case of cysteine proteases, derivatives with ketones activated by CF3 or similar groups have little or no activity (M. R. Angelastro et al., J. Med. Chem. 1990, 33, 11-13). To date only ketone derivatives in which, on the one hand, leaving groups in the a position cause irreversible inhibition and, on the other hand, the keto group is activated by a carboxylic acid derivative have been found to be effective inhibitors of calpain (see M. R. Angelastro et al., see above; WO 92/11850; WO 92,12140; WO 94/00095 and WO 95/00535). However, many of these inhibitors are derived from peptides (Zhaozhao Li et al., J. Med. Chem. 1993, 36, 3472-80; S. L. Harbenson et al., J. Med. Chem. 1994, 37, 2918-29 and see above M. R. Angelastro et al.).
Ketone derivatives which have a hetero group in the xcex1 position have also been described as calpain inhibitors. Thus, sulfur derivatives (see EP 603873) and oxygen derivatives (see WO 95/15749 and R. E. Dolle et al., J. Med. Chem. 1995, 38, 220-222) in which these hetero atoms are in the position xcex1 to the ketone are known. Ketones which have an amino or amido group in the a position are likewise known, but usually in structures derived from peptides. Thus, EP 603873 has mentioned xcex1-amino radicals carrying a heterocycle. xcex1-Amides have likewise been described several times: D. L. Flynn et al. J. Am. Chem. Soc. 1997, 119, 4874-4881; S. Natarajan et al., J. Enzym. Inhib. 1988, 2, 91-97; J. D. Godfrey et al., J. Org. Chem. 1986, 51, 3073-3075; GB 2170200; EP 159156; EP 132304; U.S. Pat. No. 4,470,973 and JP 59033260. Most of the derivatives described therein are substituted on the amide residue by other amino acid derivatives. However, the amide 
has likewise been described by D. L. Flynn et al. (see above). On the other hand, no derivatives in which the benzamide group has a substituent are mentioned. In addition, most of the compounds have been postulated as inhibitors of angiotensin converting enzyme.
An analogous sulfonamide but once again without substitution on the benzamide fragment has been described in R. F. Meyer et al., J. Med. Chem. 1982, 25, 996-996 [sic], also as inhibitor of angiotensin converting enzyme. JP 06035142 (CA 121, 267626) has described benzamide derivatives analogous to the general structure I as photographic material, although heterocycles such as hydantoins or other groups sensitive to oxidation reactions stand in R1.
The novel compounds of the general formula I in which the substitutions on the benzamide and in the position xcex1 to the keto group play important parts, with an amido or sulfonamido group being in the a position, have not previously been described and are accordingly novel.
In a number of therapies, such as [lacuna] stroke, the active ingredients are administered intravenously, for example as infusion solution. To do this it is necessary to have available substances, in this case calpain inhibitors, which have adequate solubility in water so that an infusion solution can be prepared. Many of the described calpain inhibitors have, however, the disadvantage that they have only low or no solubility in water and thus are unsuitable for intravenous administration. Active ingredients of this type can be administered only with ancillary substances intended to confer solubility in water (cf. R. T. Bartus et al. J. Cereb. Blood Flow Metab. 1994, 14, 537-544). These ancillary substances, for example polyethylene glycol, often have side effects, however, or are even incompatible. A non-peptide calpain inhibitor which is soluble in water without ancillary substances would thus be a great advantage. Such inhibitors have scarcely been described previously, and would thus show particular advantages.
Benzamide derivatives are described in the present invention. These compounds are novel and a number of derivative surprisingly show the possibility of obtaining potent non-peptide inhibitors of cysteine proteases, such as, for example, calpain, by incorporating rigid structural fragments. In addition, all the present compounds of the general formula I have at least one aliphatic amine radical and are thus able to bond [sic] salts with acids. This results in improved solubility in water and thus the compounds show the required profile for intravenous administration as is necessary, for example, for stroke therapy.
The present invention relates to substituted benzamides of the general formula I 
and their tautomeric forms, possible enantiomeric and diastereomeric forms, E and Z forms, and possible physiologically tolerated salts, in which the variables have the following meanings:
R1xe2x80x94C1-C6-alkyl, branched or unbranched, where one of the C atoms in this chain may be substituted by a phenyl ring, cyclohexyl ring, indolyl ring and an SCH3 group, and the phenyl ring in turn is substituted by by [sic] a maximum of two R4 radicals, where R4 hydrogen, C1-C4-alkyl, branched or unbranched, xe2x80x94Oxe2x80x94C1-C4-alkyl, OH, Cl, F, Br, I, CF3, NO2, NH2, CN, COOH, COOxe2x80x94C1-C4-alkyl, NHCOxe2x80x94C1-C4-alkyl, and
R2 can be NR5COxe2x80x94R6 and NHR5SO2xe2x80x94R6, and
R3 is chlorine, bromine, fluorine, C1-C6-alkyl, NHCOxe2x80x94C1-C4-alkyl, NHSO2xe2x80x94C1-C4-alkyl, NO2, xe2x80x94Oxe2x80x94C1-C4-alkyl, CN, COOH, CONH2, COOxe2x80x94C1-C4-alkyl, SO2xe2x80x94C1-C4-alkyl, xe2x80x94SO2Ph, SO2NHxe2x80x94C1-C4-alkyl, iodine, SO2NH2 and NH2, and
A can be aromatic rings and heteroaromatic rings such as naphthyl, quinolinyl, quinoxalyl, benzimidazolyl, benzothienyl, quinazolyl, phenyl, thienyl, imidazolyl, pyridyl, pyrimidyl and pyridazyl, it also being possible for the rings to be substituted by by [sic] R9 and up to 2 R8 radicals, and
B is a bond, xe2x80x94(CH2)mxe2x80x94, xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)oxe2x80x94, xe2x80x94(CH2)oxe2x80x94Sxe2x80x94(CH2)mxe2x80x94, xe2x80x94(CH2)Oxe2x80x94SOxe2x80x94(CH2)mxe2x80x94, xe2x80x94(CH2)oxe2x80x94SO2xe2x80x94(CH2)mxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94COxe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94(CH2)oxe2x80x94COxe2x80x94(CH2)mxe2x80x94, xe2x80x94(CH2)mxe2x80x94NHCOxe2x80x94(CH2)oxe2x80x94, xe2x80x94(CH2)mxe2x80x94CONHxe2x80x94(CH2)oxe2x80x94, xe2x80x94(CH2)mxe2x80x94NHSO2xe2x80x94(CH2)oxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94(CH2)mxe2x80x94SO2NHxe2x80x94(CH2)oxe2x80x94,
Axe2x80x94B together also 
R5 hydrogen and C1-C4-alkyl and
R6 is hydrogen, phenyl, naphthyl, C1-C6-alkyl, linear or branched, it being possible for a C atom in the chain to be substituted by a phenyl ring which itself may also be substituted by one or two R4 radicals, and
R8 can be hydrogen, C1-C4-alkyl, branched or unbranched, xe2x80x94Oxe2x80x94C1-C4-alkyl, OH, Cl, F, Br, I, CF3, NO2, NH2, CN, COOH, COOxe2x80x94C1-C4-alkyl, xe2x80x94NHCOxe2x80x94C1-C4-alkyl, phenyl, NHCO-phenyl, xe2x80x94NHSO2xe2x80x94C1-C4-alkyl, xe2x80x94NHSO2-phenyl, xe2x80x94SO2xe2x80x94C1-C4-alkyl, pyridine [sic] and SO2-phenyl,
R9 hydrogen, xe2x80x94CHR14xe2x80x94(CH2)pxe2x80x94R12 where R12 pyrrolidine [sic], morpholine [sic], piperidine [sic], hexahydroazepine [sic], homopiperazine [sic], 
xe2x80x83and R10 [lacuna] C1-C6-alkyl, branched or unbranched, and which may also carry a phenyl ring which is in turn substituted by by [sic] a maximum of two R11 radicals, where R11 is hydrogen, C1-C4-alkyl, branched or unbranched, xe2x80x94Oxe2x80x94C1-C4-alkyl, OH, Cl, F, Br, I, CF3, NO2, NH2, CN, COOH, COOxe2x80x94C1-C4-alkyl, NHCOxe2x80x94C1-C4-alkyl, xe2x80x94NHSO2xe2x80x94C1-C4-alkyl and xe2x80x94SO2xe2x80x94C1-C4-alkyl; and
R13 is hydrogen and C1-C6-alkyl, branched or unbranched, and
n,p is [sic], independently of one another, a number 0, 1 or 2, and
m,o is [sic], independently of one another, a number 0, 1, 2, 3 or 4.
Preferred compounds of the general formula I are those in which
A is phenyl and naphthyl, each of which may be substituted by R9, and
B is xe2x80x94SO2NHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, a bond, and xe2x80x94Cxe2x89xa1Cxe2x80x94 and
R1 ethyl, propyl, butyl and benzyl,
R2 is NHxe2x80x94SO2xe2x80x94R6 and NHxe2x80x94COxe2x80x94R6 and
R3 is hydrogen and COOR and
R6 is C1-C4-alkyl, branched and unbranched, and phenyl and
R9 hydrogen, xe2x80x94(CH2)xe2x80x94R12 where R12 pyrrolidine [sic], morpholine [sic], piperidine [sic], 
xe2x80x83and R10 C1-C6-alkyl, branched or unbranched, and
R13 can be C1-C4-alkyl, branched or unbranched.
Particularly preferred compounds of the general formula I are those in which
A is phenyl which may also be substituted by R9, and
B is xe2x80x94CHxe2x95x90CHxe2x80x94, and the B radical is in the ortho position to [sic] the benzamide of the general formula I, and
R1 butyl and benzyl
R2 is NHxe2x80x94SO2xe2x80x94R6 and
R3 is hydrogen and
R6 is C1-C4-alkyl, branched and unbranched, and phenyl and
R9 hydrogen, xe2x80x94(CH2)xe2x80x94R12 where R12 pyrrolidine [sic], morpholine [sic], piperidine [sic], 
xe2x80x83and R10 C1-C6-alkyl, branched or unbranched, and
R13 C1-C4-alkyl, branched or unbranched,
R14 can be hydrogen, methyl, ethyl.
The compounds of the formula I can be employed as racemates, as enantiomerically pure compounds or as diastereomers. If enantiomerically pure compounds are required, these can be obtained, for example, by carrying out a classical racemate resolution with the compounds of the formula I or their intermediates using a suitable optically active base or acid. On the other hand, the enantiomeric compounds can likewise be prepared by using commercially purchasable compounds, for example optically active amino acids such as phenylalanine, tryptophan and tyrosine.
The invention also relates to compounds which are mesomers or tautomers of compounds of the formula I, for example those in which the keto group in formula I is in the form of an enol tautomer.
The invention further relates to the physiologically tolerated salts of the compounds I which can be obtained by reacting compounds I with a suitable acid or base. Suitable acids and bases are listed, for example, in Fortschritte der Arzneimittelforschung, 1966, Birkhxc3xa4user Verlag, Vol. 10, pp. 224-285. These include, for example, hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid etc., and sodium hydroxide, lithium hydroxide, potassium hydroxide and tris.
The novel compounds of the general formula I can be prepared in various ways as described hereinafter (see scheme 1).
A benzoic acid II, which, where appropriate, [lacuna] simply from analogous esters by hydrolysis with acids such as hydrochloric acid, or bases such as lithium hydroxide or sodium hydroxide, in aqueous solutions or water/solvent mixtures, such as water/alcohols or water/tetrahydrofuran, at room temperature or elevated temperature, up to the boiling point of the solvent, are [sic] reacted with appropriate amino alcohols III to give the benzamides IV. This entails use of conventional peptide coupling methods which are detailed either in C. R. [sic] Larock, Comprehensive Organic Transformations, VCH Publisher, 1989, page 972 et seq., or in Houben-Weyl, Methoden der organischen Chemie, 4th edition, E5, Chapter V. It is preferred to use xe2x80x9cactivatedxe2x80x9d acid derivatives of II, with the acid group COOH being converted into a COL group. L is a leaving group such as, for example, C1, imidazole and N-hydroxybenzotriazole. This activated acid is subsequently reacted with amines to give the amides IV. The reaction takes place in anhydrous inert solvents such as methylene chloride, tetrahydrofuran and dimethylformamide at temperatures from xe2x88x9220 to +40xc2x0 C.
The amino alcohols III are prepared from analogous alcohols VII (for general method of synthesis, see: J. C. Barrish et al., J. Med. Chem. 1994, 37, 1758-1768). This entailed reacting VII, analogous to the above, with acids or sulfonic acids to give the corresponding amides or sulfonamides VIII. The protective group Z, which is usually BOC or Cbz, are [sic] then eliminated. This entails the use of conventional procedures, for example with BOC acids such as trifluoroacetic acid or hydrochloric acid, in solvents such as methylene chloride or mixtures of water and alcohols or tetrahydrofuran.
The alcohol derivatives IV can be oxidized to the novel aldehyde [sic] derivatives I. It is possible to use for this various conventional oxidation reactions (see C. R. [sic] Larock, Comprenhensive [sic] Organic Transformations, VCH Publisher, 1989, page 604 et seq.) such as, for example, Swern and Swern-analogous oxidations (T. T. Tidwell, Synthesis, 1990, 857-70), sodium hypochloride [sic]/TEMPO (S. L. Harbenson et al., see above) or Dess-Martin (J. Org. Chem. 1983, 48, 4155). These are preferably carried out in inert aprotic solvents such as dimethylformamide, tetrahydrofuran or methylene chloride with oxidizing agents such as DMSO/pyridinexc3x97SO3, DMSO/oxalyl chloride or DMSO/DCC or EDC at temperatures from xe2x88x9250 to +25xc2x0 C., depending on the method (see the above literature). 
Alternatively, an amino alcohol III can be reacted with a benzoic acid V in analogy to the linkage of II and III to give the benzamide derivative VI. In this case, Rxe2x80x2 is a functional group which then permits conversion into the AB radicals according to the invention (see below). Thus, Rxe2x80x2 in VI can be, for example, a nitro group which can subsequently be reduced catalytically in conventional ways, for example with palladium/carbon in water-soluble solvents such as alcohols, with hydrogen to give an analogous aniline (Rxe2x80x2xe2x95x90NH2). This amino group can then be converted into amides or sulfonamides. This entails the aniline being reacted with carboxylic acid or sulfonic acid derivatives in analogy to the (II+III) linkage.
Further radicals and transformation thereof can be respectively employed and carried out in analogy to the methods mentioned for preparing the AB-substituted benzoic acid derivatives.
In the cases where R3 in IV is a carboxylic ester, this can be hydrolyzed with bases and acids, for example lithium hydroxide, sodium hydroxide and hydrochloric acid, in aqueous systems or water/solvent mixtures, such as water/alcohols and water/tetrahydrofuran, to the carboxylic acid, either at room temperature or at elevated temperature (up to the boiling point of the solvent). The oxidation to I is then carried out as described above. 
Synthesis of the carboxylic esters II have [sic] already been described in some cases, or can be prepared [sic] by conventional chemical methods.
Compounds in which B is a bond are prepared by conventional aromatic coupling, for example Suzuki coupling with boric acid derivatives and halides with palladium catalysis, or copper-catalyzed coupling of aromatic halides. The alkyl-bridged radicals (B=xe2x80x94(CH2)mxe2x80x94) can be prepared by reducing the analogous ketones or by alkylating the organolithium, e.g. ortho-phenyloxazolidines [sic], or other organometallic compounds (cf. I. M. Dordor et al., J. Chem. Soc. Perkin Trans. I, 1984, 1247-52).
Ether-bridged derivatives are prepared by alkylating the corresponding alcohols or phenols with halides. The sulfoxides and sulfones can be obtained by oxidizing the corresponding thioethers. Alkene- and alkyne-bridged compounds are prepared, for example, by the Heck reaction from aromatic halides and appropriate alkenes and alkynes (cf. I. Sakamoto et al., Chem. Pharm. Bull., 1986, 34, 2754-59). The chalkones are produced by condensing acetophenones with aldehydes and can, where appropriate, be converted into the analogous alkyl derivatives by hydrogenation. Amides and sulfonamides are prepared from the amines and acid derivatives in analogy to the methods described above.
The benzamide derivatives I of the present invention are inhibitors of cysteine proteases, especially cysteine proteases such as calpains I and II and cathepsins B and L.
The inhibitory effect of the benzamides I has been determined using enzyme assays known from the literature, determining as criterion of effect a concentration of the inhibitor at which 50% of the enzyme activity is inhibited (=IC50). The amides I were measured in this way for their inhibitory effect on calpain I, calpain II and cathepsin B.
The inhibition of cathepsin B was determined by a method analogous to that of S. Hasnain et al., J. Biol. Chem., 1993, 268, 235-40. 2 xcexcl of an inhibitor solution prepared from inhibitor and DMSO (final concentrations: 100 xcexcM to 0.01 xcexcM) are added to 88 xcexcL of cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500 xcexcM buffer). This mixture is preincubated at room temperature (25xc2x0 C.) for 60 minutes and then the reaction is started by adding 10 xcexcl of 10 mM Z-Arg-Arg-pNA (in buffer with 10% DMSO). The reaction is followed in a microtiter plate reader at 405 nM [sic] for 30 minutes. The IC50s are then determined from the maximum gradients.
The testing of the inhibitory properties of calpain inhibitors takes place in buffer with 50 mM tris-HCl, pH 7.5; 0.1 M NaCl; 1 mM dithiotreithol [sic]; 0.11 mM CaCl2, using the fluorogenic calpain substrate Suc-Leu-Tyr-AMC (25 mM dissolved in DMSO, Bachem/switzerland). Human xcexc-calpain is isolated from erythrocytes, and enzyme with a purity  greater than 95%, assessed by SDS-PAGE, Western blot analysis and N-terminal sequencing, is obtained after more [sic] chromatographic steps (DEAE-Sepharose, phenyl-Sepharose, Superdex 200 and blue Sepharose). The fluorescence of the cleavage product 7-amino-4-methylcoumarin (AMC) is followed in a Spex Fluorolog fluorimeter at xcexex=380 nm and xcexem =460 nm. The cleavage of the substrate is linear in a measurement range of 60 min., and the autocatalytic activity of calpain is low, if the tests are carried out at temperatures of 12xc2x0 C. The inhibitors and the calpain substrate are added to the test mixture as DMSO solutions, and the final concentration of DMSO ought not to exceed 2%.
In a test mixture, 10 xcexcl of substrate (250 xcexcM final) and then 10 xcexcl of xcexc-calpain (2 xcexcg/ml final, i.e. 18 nM) are added to a 1 ml cuvette containing buffer. The calpain-mediated cleavage of the substrate is measured for from 15 to 20 min. Then 10 xcexcl of inhibitor (from 50 to 100 xcexcM solution in DMSO) are added and the inhibition of cleavage is measured for a further 40 min. Ki values are determined using the classical equation for reversible inhibition:
Ki=I(v0/vi)xe2x88x921;
where I=inhibitor concentration,
v0=initial rate before addition of the inhibitor;
vi=reaction rate at equilibrium.
The rate is calculated from v=AMC liberation/time, i.e. height/time.
On testing 3(2-naphthylsulfonamido)-N(3(S)-4-phenyl-1-phenylsulfonamidobutan-2-on-3-yl)benzamide [sic] (Example 1), an inhibition of more than 50% of calpain I was found at a concentration of 1 xcexcM, and thus the Ki for Example 1 is  less than 1 xcexcM.
Calpain is an intracellular cysteine protease. Calpain inhibitors must pass through the cell membrane in order to prevent intracellular proteins being broken down by calpain. Some known calpain inhibitors, such as, for example, E 64 and leupeptin, cross cell membranes only poorly and accordingly show only a poor effect on cells, although they are good calpain inhibitors. The aim is to find compounds better able to cross membranes. Human platelets are used to demonstrate the ability of calpain inhibitors to cross membranes.
Calpain-mediated breakdown of tyrosine kinase pp60src in platelets
Tyrosine kinase pp60src is cleaved by calpain after activation of platelets. This has been investigated in detail by Oda et al. in J. Biol. Chem., 1993, 268, 12603-12608. This revealed that the cleavage of pp60src can be prevented by calpeptin, a calpain inhibitor. The cellular efficacy of our substances was tested based on this publication. Fresh, citrated, human blood was centrifuged at 200 g for 15 min. The platelet-rich plasma was pooled and diluted 1:1 with platelet buffer (platelet buffer: 68 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2xc3x976 H2O, 0.24 mM NaH2PO4xc3x97H2O, 12 mM NaHCO3, 5.6 mM glucose, 1 mM EDTA, pH 7.4). After a centrifugation step and washing step with platelet buffer, the platelets were adjusted to 107 cells/ml. The human platelets were isolated at RT.
In the assay mixture, isolated platelets (2xc3x97106) were preincubated with various concentrations of inhibitors (dissolved in DMSO) at 37xc2x0 C. for 5 min. The platelets were then activated with 1 xcexcM ionophore A23187 and 5 mM CaCl2. After incubation for 5 min., the platelets were briefly centrifuged at 13000 rpm, and the pellet was taken up in SDS sample buffer (SDS sample buffer: 20 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 5 xcexcg/ml leupeptin, 10 xcexcg/ml pepstatin, 10% glycerol and 1% SDS). The proteins were fractionated in a 12% gel, and pp60src and its 52 kDa and 47 kDa cleavage products were identified by Western blotting. The polyclonal rabbit antibody used, anti-Cys-src (pp60c-rc), was purchased from Biomol Feinchemikalien (Hamburg). This primary antibody was detected using a second, HRP-coupled goat antibody (Boehringer Mannheim, FRG). The Western blotting was carried out by known methods.
The cleavage of pp60src was quantified by densitometry, using as controls unactivated (control 1: no cleavage) and ionophore- and calcium-treated platelets (control 2: corresponds to 100% cleavage). The ED50 corresponds to the concentration of inhibitor at which the intensity of the color reaction is reduced by 50%.
The test was carried out as in Choi D. W., Maulucci-Gedde M. A. and Kriegstein A. R., xe2x80x9cGlutamate neurotoxicity in cortical cell culturexe2x80x9d. J. Neurosci. 1989 [sic], 7, 357-368. The cortex halves were dissected out of 15-day old mouse embryos and the single cells were obtained enzymatically (trypsin). These cells (glia and cortical neurones) are seeded out in 24-well plates. After three days (laminin-coated plates) or seven days (ornithine-coated plates), the mitosis treatment is carried out with FDU (5-fluoro-2-deoxyuridines [sic]). 15 days after preparation of the cells, cell death is induced by adding glutamate (15 minutes). After removal of glutamate, the calpain inhibitors are added. 24 hours later, the cell damage is estimated by determining lactate dehydrogenase (LDH) in the cell culture supernatant.
It is postulated that calpain is also involved in programmed cell death (M. K. T. Squier et al., J. Cell. Physiol. 1994, 159, 229-237; T. Patel et al. Faseb Journal 1996, 590, 587-597). For this reason, in another model, cell death was induced in a human cell line with calcium in the presence of a calcium ionophore. Calpain inhibitors must get inside the cell and inhibit calpain there in order to prevent the induced cell death.
Cell death can be induced in the human cell line NT2 by calcium in the presence of the ionophore A 23187. 105 cells/well were plated out in microtiter plates 20 hours before the test. After this period, the cells were incubated with various concentrations of inhibitors in the presence of 2.5 xcexcM ionophore and 5 mM calcium. 0.05 ml of XTT (Cell Proliferation Kit II, Boehringer Mannheim) was added to the reaction mixture after 5 hours. The optical density was determined approximately 17 hours later, in accordance with the manufacturer""s information, in an SLT Easy Reader EAR 400. The optical density at which half the cells have died is calculated from the two controls with cells without inhibitors incubated in the absence and presence of ionophore.
Elevated glutamate activities occur in a number of neurological disorders or psychological disturbances and lead to states of overexcitation or toxic effects in the central nervous system (CNS). The effects of glutamate are mediated by various receptors. Two of these receptors are classified, in accordance with the specific agonists, as NMDA receptor and AMPA receptor. Antagonists to these glutamate-mediated effects can thus be employed for treating these disorders, in particular for therapeutic use for neurodegenerative disorders such as Huntington""s chorea and Parkinson""s disease, neurotoxic impairments after hypoxia, anoxia, ischemia and after lesions like those occurring after stroke and trauma, or else as antiepileptics (cf. Arzneim. Forschung 1990, 40, 511-514; TIPS, 1990, 11, 334-338; Drugs of the Future 1989, 14, 1059-1071).
Intracerebral administration of excitatory amino acids (EAA) induces such drastic overexcitation that it leads to convulsions and death of the animals (mice) within a short time. These signs can be inhibited by systemic, e.g. intraperitoneal, administration of centrally acting substances (EAA antagonists). Since excessive activation of EAA receptors in the central nervous system plays a significant part in the pathogenesis of various neurological disorders, it is possible to infer from the detected EAA antagonism in vivo that the substances may have therapeutic uses for such CNS disorders. As a measure of the efficacy of the substances, an ED50 was determined, at which 50% of the animals are free of signs, owing to the previous i.p. administration of the test substance, by means of a fixed dose of either NMDA or AMPA.
The benzamide derivatives I are inhibitors of cysteine derivatives [sic] like calpain I and II and cathepsin B and L, and can thus be used to control diseases associated with an elevated activity of calpain enzymes or cathepsin enzymes. The present amides I can accordingly be used to treat neurodegenerative disorders occurring after ischemia, trauma, subarachnoid hemorrhages and stroke, and neurodegenerative disorders such as multi-infarct dementia, Alzheimer""s disease, Huntington""s disease and epilepsies and, in addition, to treat damage to the heart after cardiac ischemia and damage due to reperfusion after vascular occlusions, damage to the kidneys after renal ischemia, skeletal muscle damage, muscular dystrophies, damage arising through proliferation of smooth muscle cells, coronary vasospasms, cerebral vasospasms, cataracts of the eyes, restenosis of the blood vessels after angioplasty. In addition, the amides I may be useful in the chemotherapy of tumors and metastasis thereof and for treating diseases in which an elevated interleukin-1 level occurs, such as inflammations and rheumatic disorders.
The pharmaceutical preparations according to the invention comprise a therapeutically effective amount of the compounds I in addition to conventional pharmaceutical ancillary substances. The active ingredients can be present in the usual concentrations for local external use, for example in dusting powders, ointments or sprays. As a rule, the active ingredients are present in an amount of from 0.001 to 1% by weight, preferably 0.001 to 0.1% by weight.
For internal use, the preparations are administered in single doses. From 0.1 to 100 mg are given per kg of body weight in a single dose. The preparation may be administered in one or more doses each day, depending on the nature and severity of the disorders.
The pharmaceutical preparations according to the invention comprise, apart from the active ingredient, the customary excipients and diluents appropriate for the required mode of administration. For local external use it is possible to use pharmaceutical ancillary substances such as ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glyco [sic] stearate, ethoxylated fatty alcohols, liquid paraffin, petrolatum and wool fat. Suitable examples for internal use are lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone.
It is also possible for antioxidants such as tocopherol and butylated hydroxyanisole, and butylated hydroxytoluene, flavor-improving additives, stabilizers, emulsifiers and lubricants to be present.
The substances which are present in the preparation in addition to the active ingredient, and the substances used in producing the pharmaceutical preparations, are toxicologically acceptable and compatible with the active ingredient in each case. The pharmaceutical preparations are produced in a conventional way, for example by mixing the active ingredient with other [sic] customary excipients and diluents.
The pharmaceutical preparations can be administered in various ways, for example orally, parenterally, such as intravenously by infusion, subcutaneously, intraperitoneally and topically. Thus, possible presentations are tablets, emulsions, solutions for infusion and injection, pastes, ointments, gels, creams, lotions, dusting powders and sprays.